JP2002010800A - Measurement of bioluminescence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and sensitive method for the measurement of bioluminescence. SOLUTION: This invention relates to (1) a method for the measurement of bioluminescence comprising the contact of a specimen with a bioluminescent reagent (luciferin, luciferase and magnesium ion) and an ATP regeneration enzyme (pyruvate orthophosphate dikinase or phosphoenol pyruvate synthetase or a mixture of adenylate kinase and pyruvate kinase) followed by the exposure of a photosensitive material with the generated bioluminescence, (2) a method for the determination of ATP using the ATP regeneration enzyme, (3) a method for the measurement of bioluminescence comprising the 1st step to contact magnesium ion, a specimen, luciferin, luciferase, pyruvate orthophosphate dikinase, phosphoenol pyruvate and pyrophosphoric acid with each other and the 2nd step to expose a photosensitive material with the generated bioluminescence and (4) a method described in the item 1 wherein the photosensitive material is photographic film, instant photographic film or photographic printing paper.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring bioluminescence.

[0002]

2. Description of the Related Art Conventionally, adenosine triphosphate (hereinafter referred to as "ATP") in the presence of magnesium ions.
It is known that luminescence occurs when luciferin and luciferase derived from insects react with luciferin. this is,
This principle is called a bioluminescence reaction.
Methods for measuring TP, methods for measuring the number of cells by measuring intracellular ATP, and the like have been developed. In addition, many reagents involved in bioluminescence (hereinafter referred to as "bioluminescent reagents") such as luciferin and luciferase have been developed.

Further, various methods have been developed for measuring a specific substance in a sample using a bioluminescent reagent. A method of mixing a sample and a bioluminescent reagent in a test tube or cell, a method of adding a bioluminescent reagent after extracting ATP from cells captured on a filtration membrane, and bringing a carrier on which a bioluminescent reagent is immobilized into contact with a sample. Method. Further, a method has been developed in which a band of a protein or nucleic acid separated by electrophoresis is detected using a bioluminescent reagent as a probe. In these measurement methods, it is an issue to measure the generated bioluminescence simply and with high sensitivity.

ATP in a reaction system, for example, AT in a sample
When the amount of P or the amount of ATP contained in the cells is small, the conventional bioluminescent reagents and methods may not provide sufficient measurement results. As a method for improving the sensitivity of a measuring method using a bioluminescent reagent, a method of adding pyruvate orthophosphate dikinase (hereinafter referred to as “PPDK”) to a reaction system is known (Japanese Patent Application Laid-Open No. 9-234099, Japanese Patent Application Laid-Open No. 11-69994). When ATP, luciferin and luciferase react, ATP is consumed, and the luminescence rapidly attenuates. With this bioluminescence reaction, AMP and pyrophosphate are generated. Since PPDK catalyzes the reaction of regenerating ATP by acting on these products in the presence of magnesium ions (ATP regeneration reaction), the luminescence in the bioluminescence reaction is not attenuated and the luminescence can be maintained for a long time. it can. For this reason, the measurement sensitivity can be improved.

[0005] In addition to PPDK, phosphoenol pyruvate synthetase is known as an enzyme that acts on a substance produced when ATP is consumed in a bioluminescence reaction to regenerate ATP (hereinafter referred to as "ATP regenerating enzyme"). It is. A method for regenerating ATP using a combination of adenylate kinase and pyruvate kinase is also known. Thus, the method of combining the bioluminescent reagent and the ATP regenerating enzyme is useful for improving the measurement sensitivity.

When measuring bioluminescence, a luminometer such as a luminescence reader BL manufactured by Aloka is usually used.
R-201, Lumitester C-100 manufactured by Kikkoman Corporation or the like is used. In addition, a method for measuring a microorganism captured on a filtration membrane with high sensitivity using a bioluminescent reagent, PPDK, and an image analysis system device (ARGUS-50 / CL manufactured by Hamamatsu Photonic Co., Ltd.) is also known (Japanese Unexamined Patent Application Publication No. Hei 11-1999). No. 69994). As another method, a method of measuring bioluminescence by exposing a photographic film to light is known, and an apparatus for carrying out the method has been developed (Japanese Patent Application Laid-Open No. 2000-2000).
No. 500568, US Pat. Nos. 3,666,631 and 5,188,965).
The method using a photographic film is also used for measuring a luminescence reaction other than bioluminescence (Japanese Patent Laid-Open No. 3-351).
No. 47), which is excellent in that an expensive measuring device such as a luminometer is not required. Regarding the method using a photographic film, higher sensitivity is required.

[0007]

An object of the present invention is to provide a simple and highly sensitive method for measuring bioluminescence.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on the above-mentioned problems, and as a result, have found that a bioluminescent reagent and A
By combining a bioluminescence reaction system using a TP regenerating enzyme and a method using a photosensitive substrate such as a photographic film, it has been found that bioluminescence can be measured simply and with high sensitivity. The present invention has been completed based on this finding.

That is, the present invention relates to the following method. (1) A method for measuring bioluminescence, which comprises exposing bioluminescence to a photosensitive substrate when measuring bioluminescence generated by contacting a sample, a bioluminescence reagent, and an ATP regenerating enzyme. (2) the biological bioluminescent reagent contains luciferin, luciferase, and magnesium ion; (1)
The method for measuring bioluminescence described. (3) The method for measuring bioluminescence according to (1), wherein the ATP-regenerating enzyme is pyruvate orthophosphate dikinase or phosphoenolpyruvate synthetase, or a mixture of adenylate kinase and pyruvate kinase. (4) A method for measuring bioluminescence including the following steps. First step: a step of contacting a sample, luciferin, luciferase, pyruvate orthophosphate dikinase, phosphoenolpyruvate and pyrophosphate in the presence of magnesium ions. Second step: The second step of exposing the bioluminescence generated in the first step to a photosensitive substrate. (5) The method for measuring bioluminescence according to any one of (1) to (4), wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring bioluminescence according to the present invention is characterized in that, when measuring bioluminescence generated by contacting a sample, a bioluminescent reagent and an ATP regenerating enzyme, the bioluminescence is exposed to a photosensitive substrate. And

1. Method for Generating Bioluminescence In the method of the present invention, a sample, a bioluminescent reagent and an AT
The method of contacting the P-regenerating enzyme is not limited, and a known method can be used. As a sample, for example, food and drink, medicine,
Examples include cosmetics, seawater, river water, industrial water, sewage, soil, urine, feces, blood, sputum, pus, cultures of cells, and collections obtained by wiping off deposits on mechanical instruments. In addition, the above sample is prepared using a suitable solvent (for example, distilled water, physiological saline,
Phosphate buffer, Tris buffer, sodium acetate buffer, etc.). In the present invention, a filter membrane capturing cells such as microorganisms may be used as a sample.

As the bioluminescent reagent, for example, a reagent containing luciferin, luciferase, and magnesium ion can be used. In this case, as the ATP regenerating enzyme, for example, PPDK and phosphoenolpyruvate synthetase can be used. Further, two or more enzymes involved in the ATP regeneration reaction, such as a mixture of adenylate kinase and pyruvate kinase, may be used in combination as an ATP regeneration enzyme. The bioluminescent reagent may contain other reagents necessary for the bioluminescent reaction and the ATP regeneration reaction, or reagents (preservatives, pH adjusters, antioxidants, etc.) for allowing each reaction to proceed stably. Good.

As PPDK, any enzyme can be used as long as it is an enzyme that acts on AMP and pyrophosphate to generate ATP. For example, it can be derived from corn leaves [Biochem.
istry 12, 2862-2867 (1973)] and sugar cane leaves [The
Biochemical Journal 114, 117-125 (1969)], and examples of microorganism-derived enzymes include, for example, Propionibacterium shermanii [Bi
ochemistry 10, 721-729 (1971)], genus Microbispora [for example, Microbispor thermoserosa (Microbispor
a thermorosea) IFO 14047].

As the luciferin and luciferase, those derived from, for example, insects (Genji firefly, Heike firefly, North American firefly, Japanese click beetle, glow firefly) can be used.

[0015] PPDK and luciferase are added to one or more amino acids in the amino acid sequence of a natural enzyme purified from cultures or tissues of the above organisms, a recombinant enzyme prepared by a genetic engineering technique, and the natural enzyme. Mutant enzymes into which mutations such as deletion, substitution, etc. have been introduced can be used. An example of a specific method for generating bioluminescence by contacting a sample, a bioluminescent reagent and an ATP regenerating enzyme includes, in the presence of magnesium ions, a sample,
The method includes contacting luciferin, luciferase, pyruvate orthophosphate dikinase, phosphoenolpyruvate and pyrophosphate.

2. Photosensitive substrate In the present invention, bioluminescence is exposed to the photosensitive substrate. As the photosensitive substrate, a material in which a material whose physical properties change by light irradiation is fixed on a suitable carrier can be used. As the photosensitive substrate, any material having a characteristic capable of sensitizing bioluminescence can be used. For example, photosensitive films such as photographic films, photographic paper, photosensitive resin, CCD (Charge Cou
pled Divice) can be used. 3. Measuring method using photographic film or photographic paper Photographic film or photographic paper is easily available as a commercial product having various characteristics, and is excellent in operability, portability, and economic efficiency. It is suitable as. As the photographic film, a high-sensitivity film for black-and-white photographs or color photographs, a high-sensitivity X-ray film, a negative type and a positive type film, and the like can be used. Hereinafter, an example of a method for exposing bioluminescence to a photographic film or photographic paper (hereinafter referred to as “photographic film or the like”) will be described.

First, a filter, a carrier, and the like (hereinafter, referred to as "test tubes, etc.") capturing test tubes, cells, and cells that generate bioluminescence are brought into contact with or close to a photographic film or the like. Both may be brought into direct contact with each other, or may be brought into contact via a thin glass plate, a plastic plate, or the like. Similar results can be obtained by photographing the test tube or the like in a dark room with a camera loaded with a photographic film. It is preferable that the photosensitive operation is performed in a dark room or a dark box.

When a photographic film is used, after exposure,
The film is developed and fixed, and if necessary, printed on photographic paper. When photographic paper is used, development and fixing are performed. The resulting bioluminescent image appears as a silhouette, point, band, etc. of the container. By observing these, it becomes possible to calculate the amount of ATP in the sample, the number of cells, and the like.
The sensitivity (ISO speed) and type of the photographic film to be used, the exposure time, the aperture and shutter speed of the camera, and the like may be appropriately set according to the type of the sample, the type of the bioluminescent reagent, the instrument used, and the like. As the photographic film, those having an ISO speed of 3000 or more can be preferably used.

4. Measuring Method Using Instant Film Among photographic films, instant films are particularly useful in that they can save the labor of development. Product Name: Instant B & W Film FP-3
000B (manufactured by Fuji Photo Film Co., Ltd.), trade name: Polaroid Coaterless Black and White Instant Pack Pack Film Type 667 (manufactured by Polaroid Co., Ltd.) and the like can be used. Hereinafter, an example of a method for exposing bioluminescence to an instant film will be described.

First, the instant film pack is mounted on an instant film holder (eg, Polaroid). After bringing a test tube or the like that emits bioluminescence into contact with the film holder with the shutter cover closed as much as possible, open the shutter cover in a dark room. After the film is exposed to bioluminescence at an appropriate time, the shutter cover is closed. Remove the film from the film holder.

5. Measurement method using CCD CCD is widely used as a photosensitive substrate in digital cameras, and is capable of storing light information as a digital signal, so that it is excellent in processing, recording, and transmitting measurement results. When a CCD is used as the photosensitive substrate, the bioluminescence can be exposed according to a method using a photographic film or the like. That is, a test tube or the like generating bioluminescence is brought into direct or indirect contact with the CCD.
Alternatively, the test tube or the like may be photographed using a digital camera using a CCD. The method of using a digital camera is excellent in that an obtained image can be immediately confirmed on a built-in monitor screen or a connected computer monitor.

6. Advantages of the present invention Method for measuring bioluminescence generated by a bioluminescent reagent containing an ATP regenerating enzyme using a luminometer (conventional method)
The advantages of the method (the present invention) of measuring using a photographic film or a photographic paper as compared with are as follows. (1) No expensive luminometer is required. (2) The measurement result can be stored in a state where it can be seen as a photograph. (3) The equipment and instruments used are compact and excellent in portability. (4) Electric energy is not required for measurement. Also,
Compared to the method (conventional method) of measuring bioluminescence generated only by a bioluminescent reagent using a photographic film or photographic paper, ATP
Advantages of the method (the present invention) for measuring bioluminescence generated by a bioluminescent reagent containing a regenerating enzyme using a photographic film or photographic paper include the following. (5) Since bioluminescence lasts longer than in the conventional method, a photographic film or the like can be sufficiently exposed, and a clear image can be obtained. Therefore, it is possible to increase the sensitivity of the measurement.

7. Application Examples of the Present Invention The present invention is particularly useful as a method for counting the number of living cells in a sample. An example of a method for counting the number of cells is described below. First,
After a sample suspected of containing cells is liquefied, it is filtered through a filtration membrane. Next, in order to extract ATP from the cells, the filtration membrane is brought into contact with an ATP extractant (such as a surfactant). Next, the filter membrane, the ATP regenerating enzyme, and the bioluminescent reagent are brought into contact with each other, and the resulting bioluminescence is exposed to the photosensitive substrate. When cells are present on the filtration membrane, the cells are observed on the photosensitive substrate as light spots. As described above, the number of living cells in the sample can be counted. Cells to be measured include fungal, yeast and bacterial cells in addition to animal and plant cells. 8. Apparatus for carrying out the present invention When carrying out the method of the present invention, known apparatuses and instruments for measuring bioluminescence or chemiluminescence using a photographic film or the like can be used as they are or partially modified. is there.

An example of an apparatus for carrying out the method of the present invention is shown in FIGS. The apparatus shown in FIG. 1A includes a dark box-shaped main body 2 that houses a photosensitive substrate 1. A test tube or the like 3 which is a bioluminescence reaction container is inserted through an insertion port 5 provided with a lid 4. It is preferable that the dark box block light when the lid is closed. However, if the device is used in a dark room, the lid is not essential. (2) is a perspective view of the device.

FIG. 2 shows an example of the apparatus when the photosensitive substrate is an instant film. In this apparatus, a film pack 7 containing a plurality of instant films is mounted on a film holder 6. In addition, the apparatus includes a support 8 for holding a test tube or the like 3 that is producing bioluminescence,
It has a shutter cover 9 for shielding light. The support is
The structure which can hold several test tubes etc. may be sufficient. After the exposure, the instant film 10 is extracted from the outlet 11. (2) is a perspective view of the device. In addition,
As shown in (3), if a light-shielding cover 12 is provided, it can be used in places other than a dark room.

FIG. 3 shows an example of the apparatus when the photosensitive substrate is a CCD. The apparatus comprises a dark box-shaped main body 2 for accommodating a CCD 13, and an insertion port 5 (with a lid 4) for inserting a test tube 3 or the like, which is a bioluminescence reaction container. CCD
Reads bioluminescent light information and converts it into electrical signals. The electric signal is output to the data output device 14 by a computer,
The image is output to a peripheral device such as a printer and reproduced as an image. Other devices necessary for collecting, digitizing, outputting, and reproducing optical information are appropriately installed. Other devices include, for example, microlenses, A / D converters, CDS (cor
elation doublesampling).

As shown in FIG. 4A, the apparatus for carrying out the present invention is provided with a condenser lens 16 in order to collect bioluminescent light 15 on the photosensitive substrate 1 and improve the measurement sensitivity. You may. When a bioluminescence reaction is caused in a test tube or cell, a structure 17 in which a part of the test tube or cell functions as a condenser lens may be used as shown in (2).

It is desirable that the surface of the test tube or cell other than the film contact surface used in the present invention is subjected to a process for preventing light scattering. The distance between a photosensitive substrate such as a photographic film and a test tube where bioluminescence is generated is preferably set to be as short as possible, and the two may be in contact with each other.

[0029]

The present invention will be described below with reference to examples. 1. Preparation of Reagent (1) Bioluminescent Reagent Containing ATP Regenerating Enzyme In order to carry out the present invention, a luminescent reagent containing PPDK as an ATP regenerating enzyme (“PPDK luminescent reagent”) was prepared. PPDK luminescent reagent is a bioluminescent reagent such as Heike firefly luciferase, luciferin, magnesium ion, and ATP-regenerating enzyme derived from microbispora thermorosa.
Pyrophosphoric acid as a reagent necessary for DK and ATP regeneration reaction,
Contains phosphoenol pyruvate, benzalkonium chloride as a reagent necessary for ATP extraction from microorganisms, and a protective agent. Its composition is as follows: Composition of PPDK luminescence reagent: 0.5 mM luciferin (Sigma) 0.27 mg / ml Mutant Heike firefly luciferase (Kikkoman) 0.13 mg / ml PPDK (from Microbispora thermorosea) 0.2 mM PPi K (potassium pyrophosphate) 1.4 mM PEP K (potassium phosphoenolpyruvate) 6.67 mM Mg Ac 4H ₂ O (magnesium acetate tetrahydrate) 0.01% benzalkonium chloride Protectant (0.06% BSA, 20 mM Tricine ( pH7.8), 1 mM EDTA
(2Na, 0.023 mM DTT) (2) Bioluminescent reagent of comparative example The following two types of bioluminescent reagents for comparison were used. As a normal sensitivity reagent: Lucifer 250 Plus (Kikkoman) As a high sensitivity reagent: Lucifer HS Plus (Kikkoman)

2. Implementation of bioluminescence reaction (1) PPDK luminescence reagent 0.01 ml ATP solution (from 2 × 10 ^-6 to 2
(10-fold dilution series to × 10 ⁻¹³ M) was added, and the reaction solution was mixed by pipetting to generate bioluminescence. (2) Bioluminescent reagent for comparison 0.05 ml of ATP extraction reagent was added to 0.05 ml of ATP solution (10-fold dilution series from 4 × 10 ⁻⁶ to 4 × 10 ⁻¹² M), and further 0.1 ml.
05 ml of luminescent reagent was added to generate bioluminescence.

3. Measurement of bioluminescence (1) Measurement by luminometer A test tube (trade name: Lumitube, manufactured by Kikkoman) is used as a reaction vessel for each reagent, and Lumitester K-100 is used.
(Kikkoman Co., Ltd.) to measure the amount of luminescence. (2) Measurement by photographic film Using a Nunc immunomodule module plate C8 (manufactured by Nunc) as a reaction vessel, the reaction was performed by dropping each reagent in one row. The interval between wells used for the reaction was appropriately adjusted depending on the intensity of light emission in order to prevent interference of light emission. An instant B & W film FP-3000B (Fuji Photo Film Co., Ltd.) was mounted on a film holder (Polaroid Co., Ltd.), and the shutter cover was opened in a dark room. Then
The bioluminescent wells were exposed on a film. The exposure time was 5 minutes or 15 minutes.

4. Measurement results (1) Measurement results by luminometer Table 1 shows the measurement results of the luminescence amount by the PPDK luminescence reagent and the bioluminescence reagent (Lucifer 250 Plus and HS Plus). From this, it was found that the PPDK luminescent reagent showed a luminescence amount about 50 times higher than Lucifer 250 plus and about 2.5 times higher than Lucifer HS plus. However, the PPDK luminescent reagent has a high blank value, and when the lower detection limit is determined from twice the blank value, the PPDK luminescent reagent and Lucifer 250 Plus have the same sensitivity of 2 × 10 ^-15 M, and the Lucifer HS Plus has the highest sensitivity. The sensitivity was 2 × 10 ^-16 M.

[0033]

[Table 1]

(2) Results of Measurement Using Photographic Film FIG. 5 shows the results of the exposure time of 5 minutes, and FIG. 6 shows the results of the exposure time of 15 minutes. With a 5-minute exposure time, the luminescence image of the PPDK luminescence reagent is 2 ×
The measurement was clearly possible up to 10 ^-13 mol / assay, and the sensitivity decrease from the case of using a luminometer was only 100 times. On the other hand, the lower detection limit of Lucifer 250 plus is 2 × 10 ^-11 mol / assay, and the lower detection limit of Lucifer HS plus is 2 × 10 ^-12 mol / assay, and the sensitivity decrease from using a luminometer is 10,000 times. I'm gone. That is, when luminescence measurement is performed using a photographic film, PP
It was shown that the DK luminescent reagent can measure 100 times more sensitively than Lucifer 250 plus and 10 times more sensitive than Lucifer HS plus.

This is because when the bioluminescent reagent of the comparative example was used, the amount of luminescence attenuated with the passage of time, but when the PPDK luminescent reagent was used, the luminescence continued due to the ATP regeneration reaction, and the film was sufficiently exposed. For reference, Table 2 shows the residual luminescence (%) after a certain time (5, 15 minutes) when the bioluminescent reagent or the PPDK luminescent reagent of the comparative example was used.
Is shown. The remaining light emission amount was calculated by the following equation. 100 x (light emission per unit time after a certain period of time) /
(Emission at the time when the emission per unit time is maximized immediately after the start of the emission reaction)

[Table 2]

On the other hand, when the exposure time is further extended to 15 minutes, the lower detection limit of the PPDK luminescent reagent is 2 × 10 ⁻¹⁴ mol / assay, which is a 10-fold increase in sensitivity as compared with the case of 5 minutes, ie,
The decrease in sensitivity from using a luminometer was tenfold. On the other hand, the lower detection limits of Lucifer 250 Plus and Lucifer HS Plus were not different from those in the case of 5 minutes, and the sensitivity decrease from the case of using a luminometer was 10,000 times. That is, the PPDK luminescent reagent is lucifer
It is possible to measure 1000 times more than 250 plus and 100 times more sensitive than Lucifer HS plus. The above 2. Lower detection limit of ATP (mol / assa) obtained by the methods (1) and (2)
y) are summarized in Table 3.

[0037]

[Table 3]

From the above results, in the method of measuring bioluminescence using a photographic film instead of a luminometer, the PPDK luminescent reagent is less sensitive than the bioluminescent reagent, resulting in the highest sensitivity. It was clearly shown that the measurement was possible. In particular, by prolonging the exposure time, the characteristics of the PPDK luminescent reagent and photographic film could be maximized. The method of the present invention, that is, a method of contacting a sample, a bioluminescent reagent and an ATP regenerating enzyme, and exposing the resulting bioluminescence to a photosensitive substrate,
It proved to be excellent as a method for measuring bioluminescence.

[0039]

According to the present invention, a method for easily and highly sensitively measuring bioluminescence is provided. According to the present invention, light emission can be recorded as an image on a photosensitive substrate such as a photographic film without using an expensive luminometer. When a photographic film or photographic paper is used as the photosensitive substrate, an apparatus for carrying out the present invention does not require an electric device. Therefore, it is possible to design a device that is simplified and has excellent portability.

The present invention provides a method for measuring a specific substance in a sample using a bioluminescent reagent and an ATP-regenerating enzyme.
It can be used in medical, hygiene, and test research settings. The present invention is particularly useful as a method for measuring ATP in a sample or ATP in a cell, or a method for counting the number of living cells captured on a filtration membrane.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an apparatus for carrying out the present invention.

FIG. 2 is a diagram illustrating an apparatus for carrying out the present invention;

FIG. 3 is a diagram illustrating an apparatus for carrying out the present invention.

FIG. 4 shows a part of an apparatus for carrying out the present invention.

FIG. 5: Measurement result of bioluminescence by photographic film of Example (5 minutes exposure)

FIG. 6 shows the results of measuring bioluminescence using the photographic film of the example (15 minutes exposure)

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 photosensitive base material 2 dark box-shaped main body 3 test tube or the like which is a bioluminescence reaction container 4 lid 5 insertion port 6 film holder 7 film pack 8 support for holding test tube or the like 9 shutter cover for shading 10 Instant film 11 Instant film take-out port 12 Cover for shading 13 CCD 14 Data output device 15 Bioluminescence light 16 Condensing lens 17 Structure of test tube etc. which functions as a condensing lens

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Toshinori Igarashi 250 Noda, Noda-shi, Chiba Kikkoman Corporation (72) Inventor Shigeya Suzuki 250 Noda, Noda-shi, Chiba Kikkoman Corporation (72) Inventor Seiji Murakami 250 Noda, Noda-shi, Chiba F-term within Kikkoman Co., Ltd. (reference) QR03 QR42 QR50 QR66 QS03 QS11 QS24 QS28 QS36 QX02

Claims (5)

[Claims] 1. A method for measuring bioluminescence, which comprises sensitizing bioluminescence to a photosensitive substrate when measuring bioluminescence generated by contacting a sample, a bioluminescent reagent and an ATP regenerating enzyme. 2. The method for measuring bioluminescence according to claim 1, wherein the bioluminescent reagent contains luciferin, luciferase, and magnesium ion. 3. The method for measuring bioluminescence according to claim 1, wherein the ATP-regenerating enzyme is pyruvate orthophosphate dikinase or phosphoenolpyruvate synthetase, or a mixture of adenylate kinase and pyruvate kinase. 4. A method for measuring bioluminescence, comprising the following steps. First
Step: a step of contacting a sample, luciferin, luciferase, pyruvate orthophosphate dikinase, phosphoenolpyruvate and pyrophosphate in the presence of magnesium ions. Second step: The second step of exposing the bioluminescence generated in the first step to a photosensitive substrate. 5. The method for measuring bioluminescence according to claim 1, wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper.